Cross-linked bioactive hydrogel matrices

ABSTRACT

The present invention is directed to a stabilized cross-linked hydrogel matrix comprising a first high molecular weight component and a second high molecular weight component that are covalently linked, and at least one stabilizing or enhancing agent, wherein the first high molecular weight component and the second high molecular weight component are each selected from the group consisting of polyglycans and polypeptides. This stabilized hydrogel matrix may be prepared as bioactive gels, pastes, slurries, cell attachment scaffolds for implantable medical devices, and casting or binding materials suitable for the construction of medical devices. The intrinsic bioactivity of the hydrogel matrix makes it useful as a gel or paste in multiple applications, including as a cell attachment scaffold that promotes wound healing around an implanted device, as gels and pastes for induction of localized vasculogenesis, wound healing, tissue repair, and regeneration, as a wound adhesive, and for tissue bulking.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/782,322, filed May 18, 2010 now U.S. Pat. No. 8,053,423, which is adivision of U.S. patent application Ser. No. 10/372,643, filed Feb. 21,2003, now U.S. Pat. No. 7,799,767, which claims priority to ProvisionalApplication Ser. No. 60/358,625, filed Feb. 21, 2002. All of theforegoing applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to cross-linked bioactive hydrogelmatrices that are appropriate for use in therapeutic methods based onthe induction of localized vasculogenesis, wound healing, tissue repair,and tissue regeneration.

BACKGROUND OF THE INVENTION

The replacement or repair of damaged or diseased tissues or organs byimplantation has been, and continues to be, a long-standing goal ofmedicine towards which tremendous progress has been made. Working towardthat goal, there is an increasing interest in tissue engineeringtechniques where biocompatible, biodegradable materials are used as asupport matrix, as a substrate for the delivery of cultured cells, orfor three-dimensional tissue reconstruction (Park, S., “Characterizationof porous collagen/hyaluronic acid scaffold modified by1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking”Biomaterials 23:1205-1212 (2002)). However, one of the most seriousproblems restricting the use of implanted materials is the wound healingresponse by the body elicited by the implanted foreign materials(Ratner, B. D., “Reducing capsular thickness and enhancing angiogenesisaround implant drug release systems” Journal of Controlled Release78:211-218 (2002)).

Biocompatibility is defined as the appropriate response of the host to aforeign material used for its intended application. Biocompatibilityfurther refers to the interaction between the foreign material and thetissues and physiological systems of the patient treated with theforeign material. Protein binding and subsequent denaturation as well ascell adhesion and activation have been invoked as determinants of amaterial's biocompatibility. Biocompatibility also implies that theimplant avoids detrimental effects from the host's various protectivesystems and remains functional for a significant period of time. Invitro tests designed to assess cytotoxicity or protein binding areroutinely used for the measurement of a material's potentialbiocompatibility. In other words, the biocompatibility of a material isdependent upon its ability to be fully integrated with the surroundingtissue following implantation.

Previous research has shown that the specific interactions between cellsand their surrounding extracellular matrix play an important role in thepromotion and regulation of cellular repair and replacement processes(Hynes, S. O., “Integrins: a family of cell surface receptors” Cell48:549-554 (1987)). Consequently, there has been a heightened interestin work related to biocompatible polymers useful in therapeuticapplications. One particular class of polymers that have proven usefulfor such applications, including contact lens materials, artificialtendons, matrices for tissue engineering, and drug delivery systems, ishydrogels (Schacht, E., “Hydrogels prepared by crosslinking of gelatinwith dextran dialdehyde” Reactive & Functional Polymers 33:109-116(1997)). Hydrogels are commonly accepted to be materials consisting of apermanent, three-dimensional network of hydrophilic polymers with waterfilling the space between the polymer chains. Hydrogels may be obtainedby copolymerizing suitable hydrophilic monomers, by chain extension, andby cross-linking hydrophilic pre-polymers or polymers.

Prior work has shown that a thermoreversible hydrogel matrix, which isliquid near physiologic temperatures, elicits vasculogenesis andmodulates wound healing in dermal ulcers (Usala A. L. et al. “Inductionof fetal-like wound repair mechanisms in vivo with a novel matrixscaffolding” Diabetes 50 (Supplement 2): A488 (2001), and Usala A. L. etal., “Rapid Induction of vasculogenesis and wound healing using a novelinjectable connective tissue matrix” Diabetes 49 (Supplement 1): A395(2000)). This bioactive hydrogel material has also been shown to improvethe healing in response to implanted foreign materials; demonstrating adecrease in the surrounding fibrous capsule thickness and a persistentincrease in blood supply immediately adjacent to implanted materialsexposed to this thermoreversible hydrogel. (Ravin A. G. et al., “Long-and Short-Term Effects of Biological Hydrogels on Capsule MicrovascularDensity Around Implants in Rats” J Biomed Mater Res 58(3):313-8 (2001)).However the use of such a bioactive thermoreversible hydrogel intherapeutic applications requiring three-dimensional and thermalstability is not practical because the hydrogel is molten at physiologictemperatures. Accordingly, there is a need for a bioactive material thatis stable at body temperatures and thus appropriate for use either as amedical device or in medical applications, particularly those intendedfor use in mammals. A particular biopolymer for use in medicalapplications is disclosed in U.S. Pat. No. 6,132,759, which relates to amedicament containing a biopolymer matrix comprising gelatincross-linked with oxidized polysaccharides. The biopolymer of the '759patent is claimed to be useful for treating skin wounds ordermatological disorders when a wound healing stimulating drug isincorporated therein. Similarly, U.S. Pat. No. 5,972,385 describes amatrix formed by reacting a modified polysaccharide with collagen thatmay subsequently be used for tissue repair when combined with growthfactors. Various additional publications have described polymers andco-polymers for use in medical applications, such as drug delivery,tissue regeneration, wound healing, wound dressings, adhesion barriers,and wound adhesives. (See, for example, Draye, J. P. et al., “In vitrorelease characteristics of bioactive molecules from dextran dialdehydecross-linked gelatin hydrogel films” Biomaterials 19:99-107 (1998);Draye, J. P. et al., “In vitro and in vivo biocompatibility of dextrandialdehyde cross-linked gelatin hydrogel films” Biomaterials19:1677-1687 (1998); Kawai, K. et al., “Accelerated tissue regenerationthrough incorporation of basic fibroblast growth factor impregnatedgelatin microspheres into artificial dermis” Biomaterials 21:489-499(2000); Edwards, G. A. et al., “In vivo evaluation of collagenousmembranes as an absorbable adhesion barrier” Biomed. Mater. Res.34:291-297 (1997); U.S. Pat. Nos. 4,618,490; and 6,165,488.) Suchbiocompatible polymers, however, are generally only therapeuticallyeffective when combined with other therapeutic agents, such as growthfactors, clotting factors, antibiotics, and other drugs.

Several biocompatible polymers previously known are based at least inpart on collagen or collagen derived material. Additionally, other knownbiocompatible polymers are based on polysaccharides, particularlydextran. In some instances, biopolymers have been formed bycross-linking gelatin and dextran; however, the usefulness of suchpolymers for long-term use in the body has not been shown. It is welldocumented that gelatin and dextran are incompatible in aqueous solutionmaking it difficult to produce co-polymers that are stable at bodytemperatures.

Thus, there still remains a need for stabilized, bioactive hydrogelsthat are useful for medical applications where stable, long-term use inthe body is desired.

BRIEF SUMMARY OF THE INVENTION

A stabilized cross-linked bioactive hydrogel matrix useful as atherapeutic gel or paste is provided. The viscosity of the bioactivehydrogel of the invention may be varied over a wide range by controllingthe process conditions using parameters well known to those skilled inthe art. These bioactive hydrogels may be used either as therapeuticmedical devices or as adjuvants to other forms of therapy requiring amodulation of localized wound healing and tissue integration. Thehydrogel matrices of the invention comprise a first high molecularweight component and a second high molecular weight component covalentlycross-linked to the first high molecular weight component, wherein thefirst high molecular weight component and the second high molecularweight component are each selected from the group consisting ofpolyglycans and polypeptides. The matrix further comprises one or moreenhancing agents, such as polar amino acids, amino acid analogues, aminoacid derivatives, intact collagen, and divalent cation chelators, suchas ethylenediaminetetraacetic acid (EDTA) or salts thereof. In apreferred embodiment, the composition comprises a high molecular weightpolyglycan covalently bonded to a high molecular weight polypeptide. Thetwo high molecular weight components are preferably dextran and gelatin.Preferred enhancing agents include polar amino acids, such as cysteine,arginine, lysine, and glutamic acid, EDTA or salts thereof, and mixturesor combinations thereof.

A method for preparing a stabilized cross-linked bioactive hydrogelmatrix is also provided. The method comprises providing a mixture of afirst high molecular weight component, a second high molecular weightcomponent, and an enhancing agent, and reacting the first high molecularweight component with the second high molecular weight component underconditions sufficient to covalently cross-link the first high molecularweight component to the second high molecular weight component. As wouldbe understood, the two high molecular weight components can becross-linked during or after addition of the enhancing agent(s).

In another aspect, the present invention provides a method for using astabilized cross-linked bioactive hydrogel matrix for enhancing tissueregeneration. The method comprises the steps of identifying a specificsite in need of tissue regeneration and administering a therapeuticallyeffective amount of a stabilized cross-linked bioactive hydrogel matrix,as described above, to the identified site.

In yet another aspect, a method for using a stabilized cross-linkedbioactive hydrogel matrix for adding bulk to tissue is provided. Themethod comprises the steps of identifying a specific site in need ofadded tissue bulk and administering a therapeutically effective amountof a stabilized cross-linked bioactive hydrogel matrix, as describedabove, to the identified site.

In still another aspect, the invention provides a method for preparing abone implant using a stabilized cross-linked bioactive hydrogel matrixof the present invention. The method comprises the steps of providing anamount of an osteoconductive or osteoinductive material, such as calciumaluminate, hydroxyapatite, alumina, zirconia, aluminum silicates,calcium phosphate, bioactive glass, ceramics, collagen, autologous bone,allogenic bone, xenogenic bone, coralline, or derivates or combinationsthereof, providing a stabilized cross-linked bioactive hydrogel matrixas described above, combining the osteoconductive or osteoinductivematerial with the hydrogel matrix to form a pourable and castablecomposite paste, casting the paste into a shaped mold, allowing thepaste in the shaped mold to harden, and removing the cast paste from theshaped mold.

In a further aspect of the present invention is provided a method forusing a stabilized cross-linked bioactive hydrogel matrix in combinationwith viable tissue cells for therapeutic treatment.

In another aspect of the present invention is provided a method forusing a stabilized cross-linked bioactive hydrogel matrix for treating awound, for example, as a wound covering or as a tissue sealant.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, which are not necessarily drawn toscale, and wherein:

FIG. 1 illustrates formation of open alpha chains derived from collagenmonomers;

FIG. 2A illustrates the effect of the association of the alpha chainswith dextran;

FIG. 2B illustrates the behavior of the alpha chains without associationof the dextran;

FIG. 3 illustrates the effect of other hydrogel matrix additives;

FIG. 4 illustrates an embodiment of a covalently cross-linkedgelatin/dextran matrix of the invention;

FIG. 5 illustrates graphically the effect of a hydrogel matrix inpromoting cell aggregation;

FIG. 6 illustrates the use of a cross-linked embodiment of the bioactivematrix of the present invention in bone repair; and

FIG. 7 illustrates graphically the relationship of the overall strengthof the cross-linked hydrogel matrix to the amount of dextran oxidation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Likenumbers refer to like elements throughout.

The formulation of a thermoreversible hydrogel matrix providing a cellculture medium and composition for preserving cell viability is taughtby U.S. Pat. No. 6,231,881, herein incorporated by reference in itsentirety. Additionally, a hydrogel matrix useful in promotingvascularization is provided in U.S. Pat. No. 6,261,587, hereinincorporated by reference in its entirety. The thermoreversible hydrogelmatrix taught by these references is a gel at storage temperatures andmolten at physiologic temperatures, and comprises a combination of acollagen-derived component, such as gelatin, a long chain polyglycan,such as dextran, and effective amounts of other components, such aspolar amino acids. The thermoreversible hydrogel matrix taught by thesereferences is discussed below in connection with FIGS. 1-3.

Collagen is a major protein component of the extracellular matrix ofanimals. Collagen is assembled into a complex fibrillar organization.The fibrils are assembled into bundles that form the fibers. The fibrilsare made of five microfibrils placed in a staggered arrangement. Eachmicrofibril is a collection of collagen rods. Each collagen rod is aright-handed triple-helix, each strand being itself a left-handed helix.Collagen fibrils are strengthened by covalent intra- and intermolecularcross-links which make the tissues of mature animals insoluble in coldwater. When suitable treatments are used, collagen rods are extractedand solubilized where they keep their conformation as triple-helices.This is denatured collagen and differs from the native form of collagen,but has not undergone sufficient thermal or chemical treatment to breakthe intramolecular stabilizing covalent bonds found in collagen. Whencollagen solutions are extensively heated, or when the native collagencontaining tissues are subjected to chemical and thermal treatments, thehydrogen and covalent bonds that stabilize the collagen helices arebroken, and the molecules adopt a disordered conformation. By breakingthese hydrogen bonds, the polar amine and carboxylic acid groups are nowavailable for binding to polar groups from other sources or themselves.This material is gelatin and is water-soluble at 40-45° C.

As noted above, gelatin is a form of denatured collagen, and is obtainedby the partial hydrolysis of collagen derived from the skin, whiteconnective tissue, or bones of animals Gelatin may be derived from anacid-treated precursor or an alkali-treated precursor. Gelatin derivedfrom an acid-treated precursor is known as Type A, and gelatin derivedfrom an alkali-treated precursor is known as Type B. The macromolecularstructural changes associated with collagen degradation are basicallythe same for chemical and partial thermal hydrolysis. In the case ofthermal and acid-catalyzed degradation, hydrolytic cleavage predominateswithin individual collagen chains. In alkaline hydrolysis, cleavage ofinter-and intramolecular cross-links predominates.

FIG. 1 illustrates the hydrolytic cleavage of the tropocollagen 10,forming individual polar alpha chains of gelatin 15. Heatingtropocollagen 10 disrupts the hydrogen bonds that tightly contain thetriple stranded monomers in mature collagen. FIGS. 2A-2B illustratestabilization of the matrix monomeric scaffolding by the introduction ofa long-chain polyglycan, such as dextran 20. As depicted in FIG. 2A, thedextran 20 serves to hold open the gelatin 15, that has been previouslyheated, by interfering with the natural predisposition of the gelatin 15to fold upon itself and form hydrogen bonds between its polar groups. Inthe absence of dextran 20, as shown in FIG. 2B, when the gelatin 15begins to cool, it will form hydrogen bonds between the amino andcarboxylic acid groups within the linear portion of the monomer and foldupon itself, thus limiting available sites for cellular attachment.

The thermoreversible matrix contains a polyglycan, such as dextran, at atherapeutically effective concentration ranging from, for example, about0.01 to about 10 mM, preferably about 0.01 to about 1 mM, mostpreferably about 0.01 to about 0.1 mM. In one embodiment, dextran ispresent at a concentration of about 0.09 mM.

The thermoreversible matrix also contains gelatin, at a therapeuticallyeffective concentration ranging from, for example, about 0.01 to about40 mM, preferably about 0.05 to about 30 mM, most preferably about 1 to5 mM. Advantageously, the gelatin concentration is approximately 1.6 mM.

In order to increase cell binding, intact collagen may be added in smallamounts to the thermoreversible matrix in order to provide additionalstructure for the cells contained in the matrix. The final concentrationof intact collagen is from about 0 to about 5 mM, preferably about 0 toabout 2 mM, most preferably about 0.05 to about 0.5 mM. In oneembodiment, the concentration of intact collagen is about 0.11 mM.

The thermoreversible matrix may additionally contain an effective amountof polar amino acids, which are commonly defined to include tyrosine,cysteine, serine, threonine, asparagine, glutamine, asparatic acid,glutamic acid, arginine, lysine, and histidine. For application in thepresent invention, the amino acids are preferably selected from thegroup consisting of cysteine, arginine, lysine, histidine, glutamicacid, aspartic acid and mixtures thereof, or derivatives or analoguesthereof. By amino acid is intended all naturally occurring alpha aminoacids in both their D and L stereoisomeric forms, and their analoguesand derivatives. An analog is defined as a substitution of an atom orfunctional group in the amino acid with a different atom or functionalgroup that usually has similar properties. A derivative is defined as anamino acid that has another molecule or atom attached to it. Derivativeswould include, for example, acetylation of an amino group, amination ofa carboxyl group, or oxidation of the sulfur residues of two cysteinemolecules to form cystine. The total concentration of all polar aminoacids is generally between about 3 to about 150 mM, preferably about 10to about 65 mM, and more preferably about 15 to about 40 mM.

Advantageously, the added polar amino acids comprise L-cysteine,L-glutamic acid, L-lysine, and L-arginine. The final concentration ofL-glutamic acid is generally about 2 to about 60 mM, preferably about 5to about 40 mM, most preferably about 10 to about 20 mM. In oneembodiment, the concentration of L-glutamic acid is about 15 mM. Thefinal concentration of L-lysine is generally about 0.5 to about 30 mM,preferably about 1 to about 15 mM, most preferably about 1 to about 10mM. In one embodiment, the concentration of L-lysine is about 5.0 mM.The final concentration of L-arginine is generally about 1 to about 40mM, preferably about 1 to about 30 mM, most preferably about 5 to about15 mM. In one embodiment, the final concentration of arginine is about10 mM. The final concentration of L-cysteine, which provides disulfidelinkages, is generally about 5 to about 500 μM, preferably about 10 toabout 100 μM, most preferably about 15 to about 25 μM. In oneembodiment, the final concentration of cysteine is about 20 μM.

The thermoreversible matrix is preferably based upon a physiologicallycompatible buffer, one embodiment being Medium 199, a common nutrientsolution used for in vitro culture of various mammalian cell types(available commercially from Sigma Chemical Company, St. Louis, Mo.),which is further supplemented with additives and additional amounts ofsome medium components, such as supplemental amounts of polar aminoacids as described above.

Advantageously, aminoguanidine may be added to this formulation;however, other L-arginine analogues may also be used in the presentinvention, such as N-monomethyl L-arginine, N-nitro-L-arginine, orD-arginine. The final concentration of aminoguanidine is generally about5 to about 500 μM, preferably about 10 to about 100 μM, most preferablyabout 15 to about 25 μM. In one embodiment, the final concentration isabout 20 μM.

Additionally, the matrix may include one or more divalent cationchelators, which increase the rigidity of the matrix by formingcoordinated complexes with any divalent metal ions present. Theformation of such complexes leads to the increased rigidity of thematrix by removing the inhibition of hydrogen bonding between —NH₂ and—COOH caused by the presence of the divalent metal ions. A preferredexample of a divalent cation chelator that is useful in the presentinvention is ethylenediaminetetraacetic acid (EDTA) or a salt thereof.The concentration range for the divalent cation chelator, such as EDTA,is generally about 0.01 to about 10 mM, preferably 1 to about 8 mM, mostpreferably about 2 to about 6 mM. In a one embodiment, EDTA is presentat a concentration of about 4 mM.

FIG. 3 illustrates the effect of polar amino acids and L-cysteine addedto stabilize the units 25, formed by the gelatin 15 and dextran 20, bylinking the exposed monomer polar sites to, for example, arginine'samine groups or glutamic acid's carboxylic acid groups. Furthermore,disulfide linkages can be formed between L-cysteine molecules (therebyfowling cystine), which in turn form hydrogen bonds to the gelatin 15.

The mechanical and thermal characteristics of the thermoreversiblehydrogel described above are to a large extent determined by thethermomechanical properties of one of its major components, gelatin.Gelatin-based matrices typically are molten at near physiologictemperatures and hence cannot be expected to have the requisitedurability and mechanical properties when required for implantation as amedical device in certain applications. Therefore, it is imperative tostabilize these gels through a variety of intermolecular interactionsincluding hydrogen bonding, electrostatic or polar amino acid mediatedbonding, hydrophobic bonding and covalent bonding. Although not wishingto be bound by theory, it is believed that the types of bondingmechanisms described above in association with a long chain polyglycanstabilize polypeptides such as gelatin. For example, as discussed inmore detail below, the positively charged polar groups of thecollagen-derived alpha chains are then able to associate with thenegatively charged hydroxyl groups of the repeating glucose units foundin, for example, dextran. The gelatin and dextran than a compositebioactive hydrogel containing macromolecular proteoglycan-typestructures.

Unlike the prior art thermoreversible matrix discussed above, thepresent invention provides stabilized compositions comprising across-linked bioactive hydrogel matrix that can be used, for example, topromote wound healing or vasculogenesis. The present invention is alsodirected to a method for preparing a cross-linked bioactive hydrogelmatrix that is therapeutically useful at physiological temperatures. By“bioactive” is intended the ability to facilitate or discourage acellular or tissue response of a host to foreign materials introduced tothe body. Examples include, but are not limited to, induction ofvasculogenesis, inhibition of the formation of a foreign body response,promotion of cellular attachment to the scaffold material, and promotionof tissue regeneration. The term “stabilized” or “stable” is intended torefer to compositions that are water-swellable, poorly soluble, solid orsemi-solid materials at physiological temperature (i.e., about 37° C.)and in physiological fluids (e.g., aqueous body fluids having aphysiological pH of about 7.4), which remain present in the host forsufficient time to achieve the intended response.

The stabilized bioactive hydrogel matrix of the invention is formed fromat least two high molecular weight components. The high molecular weightcomponents of the bioactive hydrogel matrix are selected from the groupconsisting of high molecular weight polyglycans, high molecular weightpolypeptides, and combinations thereof. By high molecular weightpolyglycan is intended any polysaccharide consisting of more than about10 monosaccharide residues joined to each other by glycosidic linkages.The polyglycan may consist of the same monosaccharide residues, orvarious monosaccharide residues or derivatives of monosaccharideresidues. Dextran, a preferred polysaccharide, solely comprises glucoseresidues. Dextran typically comprises linear chains of α(1→6)-linkedD-glucose residues, often with α(1→2)- or α(1→3)-branches. Nativedextran, produced by a number of species of bacteria of the familyLactobacilliaceae, is a polydisperse mixture of components.

The polyglycan component preferably has a molecular weight range ofabout 2,000 to about 8,000,000 Da, more preferably about 20,000 to about1,000,000 Da. Unless otherwise noted, molecular weight is expressedherein as number average molecular weight (M_(n)), which is defined as

$\frac{\sum{NiMi}}{\sum{Ni}},$wherein Ni is the number of polymer molecules (or the number of moles ofthose molecules) having molecular weight Mi.

Any polysaccharide, including glycosaminoglycans (GAGs) orglucosaminoglycans, with suitable viscosity, molecular mass and otherdesirable properties may be utilized in the present invention. Byglycosaminoglycan is intended any glycan (i.e., polysaccharide)comprising an unbranched polysaccharide chain with a repeatingdisaccharide unit, one of which is always an amino sugar. Thesecompounds as a class carry a high negative charge, are stronglyhydrophilic, and are commonly called mucopolysaccharides. This group ofpolysaccharides includes heparin, heparan sulfate, chondroitin sulfate,dermatan sulfate, keratan sulfate, and hyaluronic acid. These GAGs arepredominantly found on cell surfaces and in the extracellular matrix. Byglucosaminoglycan is intended any glycan (i.e. polysaccharide)containing predominantly monosaccharide derivatives in which analcoholic hydroxyl group has been replaced by an amino group or otherfunctional group such as sulfate or phosphate. An example of aglucosaminoglycan is poly-N-acetyl glucosaminoglycan, commonly referredto as chitosan. Exemplary polysaccharides that may be useful in thepresent invention include dextran, heparan, heparin, hyaluronic acid,alginate, agarose, carageenan, amylopectin, amylose, glycogen, starch,cellulose, chitin, chitosan and various sulfated polysaccharides such asheparan sulfate, chondroitin sulfate, dextran sulfate, dermatan sulfate,or keratan sulfate.

By high molecular weight polypeptide is intended any tissue-derived orsynthetically produced polypeptide, such as collagens orcollagen-derived gelatins. Although collagen-derived gelatin is thepreferred high molecular weight polypeptide component, othergelatin-like components characterized by a backbone comprised ofsequences of amino acids having polar groups that are capable ofinteracting with other molecules can be used. For example, keratin,decorin, aggrecan, glycoproteins (including proteoglycans), and the likecould be used to produce the polypeptide component. In one embodiment,the polypeptide component is porcine gelatin from partially hydrolyzedcollagen derived from skin tissue. Polypeptides derived from other typesof tissue could also be used. Examples include, but are not limited to,tissue extracts from arteries, vocal chords, pleura, trachea, bronchi,pulmonary alveolar septa, ligaments, auricular cartilage or abdominalfascia; the reticular network of the liver; the basement membrane of thekidney; or the neurilemma, arachnoid, dura mater or pia mater of thenervous system. Purified polypeptides including, but not limited to,laminin, nidogen, fibulin, and fibrillin or protein mixtures such asthose described by U.S. Pat. Nos. 6,264,992 and 4,829,000, extracts fromcell culture broth as described by U.S. Pat. No. 6,284,284, submucosaltissues such as those described in U.S. Pat. No. 6,264,992, or geneproducts such as described by U.S. Pat. No. 6,303,765 may also be used.Another example of a suitable high molecular weight polypeptide is afusion protein formed by genetically engineering a known reactivespecies onto a protein. The polypeptide component preferably has amolecular weight range of about 3,000 to about 3,000,000 Da, morepreferably about 30,000 to about 300,00 0Da.

In a preferred embodiment, gelatin and dextran are components of thebioactive matrix of the present invention. For ease of describing theinvention, the terms “gelatin” and “dextran” are used throughout withthe understanding that various alternatives as described above, such asother polyglycan and polypeptide components readily envisioned by thoseskilled in the art, are contemplated by the present invention.

In one embodiment of the present invention, as illustrated in FIG. 4,dextran 20 is covalently crosslinked to gelatin 15 by linkages 70,thereby forming a crosslinked network 50. The linkages 70 either resultfrom reaction of functional groups on the gelatin 15 with functionalgroups on the dextran 20, or result from reaction of a bifunctionalcrosslinker molecule with both the dextran 20 and gelatin 15. Asexplained in greater detail below, one method of crosslinking gelatinand dextran is to modify the dextran molecules 20, such as by oxidation,in order to form functional groups suitable for covalent attachment tothe gelatin 15. This stabilized cross-linked bioactive network 50 yieldstherapeutically useful gels and pastes that are insoluble in physiologicfluids at physiological temperatures. No additional substrate or surfaceis required. The so-formed gels and pastes are appropriate for thedevelopment of therapeutic methods based on the induction of a localizedvasculogenesis, wound healing, tissue repair, and regeneration. Suchbioactive hydrogel gels and pastes may be used, for example, to repairischemic regions of the heart or peripheral vessels, facilitate bonerepair, or to provide a localized scaffolding for wound healing andtissue repair.

In one embodiment of the method of making the cross-linked hydrogelmatrix, one of the high molecular weight components must be modified toform reactive groups suitable for cross-linking For instance, thedextran or other polyglycan component can be modified, such as byoxidation, in order to cross-link with the gelatin component. One knownreaction for oxidizing polysaccharides is periodate oxidation. The basicreaction process utilizing periodate chemistry is well known andappreciated by those skilled in the art. Periodate oxidation isdescribed generally in Affinity Chromatography: A Practical Approach,Dean, et al., IRL Press, 1985 ISBN0-904147-71-1, which is incorporatedby reference in its entirety. The oxidation of dextran by the use ofperiodate-based chemistry is described in U.S. Pat. No. 6,011,008, whichis herein incorporated by reference in its entirety.

In periodate oxidation, polysaccharides may be activated by theoxidation of the vicinal diol groups. With polyglycans, this isgenerally accomplished through treatment with an aqueous solution of asalt of periodic acid, such as sodium periodate (NaIO₄), which oxidizesthe sugar diols to generate reactive aldehyde groups (e.g. dialdehyderesidues). This method is a rapid, convenient alternative to other knownoxidation methods, such as those using cyanogen bromide. Polyglycansactivated by periodate oxidation may be stored at 4° C. for several dayswithout appreciable loss of activity.

Polyglycan materials, such as dextran, activated in this manner readilyreact with materials containing amino groups, such as gelatin, producinga cross-linked material through the formation of Schiff's base links. ASchiff base is a name commonly used to refer to the imine formed by thereaction of a primary amine with an aldehyde or ketone. The aldehydegroups formed on the cellulosic surface react with most primary aminesbetween pH values from about 4 to about 6. The Schiff s base links formbetween the dialdehyde residues of the polyglycan and the free aminogroups on the protein. The cross-linked product may subsequently bestabilized (i.e. formation of stable amine linkages) by reduction with aborohydride, such as sodium borohydride (NaBH₄) or sodiumcyanoborohydride (NaBH₃CN). The residual aldehyde groups may be consumedwith ethanolamine or other amine containing species to further modifythe cross-linked matrix. Other methods known to those skilled in the artmay be utilized to provide reactive groups on either one or both of thehigh molecular weight components of the matrix.

In the present invention, periodate chemistry is used with dextran toform a multifunctional polymer that can then react with gelatin andenhancing agents present during the manufacturing process. The periodatereaction leads to the formation of polyaldehyde polyglycans that arereactive with primary amines. For example, high molecular weightpolypeptides and high molecular weight polyglycans may form covalenthydrogel complexes that are colloidal or covalently cross-linked gels.Covalent bonding occurs between reactive groups of the dextran andreactive groups of the gelatin component. The reactive sites on thegelatin include amine groups provided by arginine, asparagine,glutamine, and lysine. These amine groups react with the aldehyde orketone groups on the dextran to form a covalent bond. These hydrogelscan be readily prepared at temperatures from about 34° C. to about 90°C. Additionally, the hydrogels can be prepared at a pH range of fromabout 5 to about 9, preferably from about 6 to about 8, and mostpreferably from about 7 to about 7.6.

By controlling the extent of dextran activation and the reaction time,one can produce stabilized biomimetic scaffolding materials of varyingviscosity and stiffness. By “biomimetic” is intended compositions ormethods imitating or stimulating a biological process or product. Somebiomimetic processes have been in use for several years, such as theartificial synthesis of vitamins and antibiotics. More recently,additional biomimetic applications have been proposed, includingnanorobot antibodies that seek and destroy disease-causing bacteria,artificial organs, artificial arms, legs, hands, and feet, and variouselectronic devices. The biomimetic scaffolding materials of the presentinvention yield therapeutically useful gels and pastes that are stableat about 37° C., or body temperature. These gels are capable ofexpansion and/or contraction, but will not dissolve in aqueous solution.

As an alternate method for forming the crosslinked dextran/gelatinnetwork, a multifunctional cross-linking agent may be utilized as areactive moiety that covalently links the gelatin and dextran chains.Such bifunctional cross-linking agents may include glutaraldehyde,epoxides (e.g., bis-oxiranes), oxidized dextran, p-azidobenzoylhydrazide, N[α-maleimidoacetoxy]succinimide ester, p-azidophenyl glyoxalmonohydrate, bis-[β-(4-azidosalicylamido)ethyl]disulfide,bis[sulfosuccinimidyl]suberate, dithiobis[succinimidyl proprionate,disuccinimidyl suberate, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride, and other bifunctional cross-linking reagents known tothose skilled in the art.

In another embodiment utilizing a cross-linking agent, polyacrylatedmaterials, such as ethoxylated (20) trimethylpropane triacrylate, may beused as a non-specific photo-activated cross-linking agent. Componentsof an exemplary reaction mixture would include a thermoreversiblehydrogel held at 39 ° C., polyacrylate monomers, such as ethoxylated(20) trimethylpropane triacrylate, a photo-initiator, such as eosin Y,catalytic agents, such as 1-vinyl-2-pyrrolidinone, and triethanolamine.Continuous exposure of this reactive mixture to long-wavelength light(>498 nm) would produce a cross-linked hydrogel network

The stabilized cross-linked hydrogel matrix of the present invention maybe further stabilized and enhanced through the addition of one or moreenhancing agents. By “enhancing agent” or “stabilizing agent” isintended any compound added to the hydrogel matrix, in addition to thehigh molecular weight components, that enhances the hydrogel matrix byproviding further stability or functional advantages. Suitable enhancingagents, which are admixed with the high molecular weight components anddispersed within the hydrogel matrix, include many of the additivesdescribed earlier in connection with the thermoreversible matrixdiscussed above. The enhancing agent can include any compound,especially polar compounds, that, when incorporated into thecross-linked hydrogel matrix, enhance the hydrogel matrix by providingfurther stability or functional advantages.

Preferred enhancing agents for use with the stabilized cross-linkedhydrogel matrix include polar amino acids, amino acid analogues, aminoacid derivatives, intact collagen, and divalent cation chelators, suchas ethylenediaminetetraacetic acid (EDTA) or salts thereof Polar aminoacids is intended to include tyrosine, cysteine, serine, threonine,asparagine, glutamine, aspartic acid, glutamic acid, arginine, lysine,and histidine. The preferred polar amino acids are L-cysteine,L-glutamic acid, L-lysine, and L-arginine. Suitable concentrations ofeach particular preferred enhancing agent are the same as noted above inconnection with the thermoreversible hydrogel matrix. Polar amino acids,EDTA, and mixtures thereof, are preferred enhancing agents. Theenhancing agents can be added to the matrix composition before or duringthe crosslinking of the high molecular weight components.

The enhancing agents are particularly important in the stabilizedcross-linked bioactive hydrogel matrix because of the inherentproperties they promote within the matrix. The hydrogel matrix exhibitsan intrinsic bioactivity that will become more evident through theadditional embodiments described hereinafter. It is believed theintrinsic bioactivity is a function of the unique stereochemistry of thecross-linked macromolecules in the presence of the enhancing andstrengthening polar amino acids, as well as other enhancing agents.

For example, aggregation of human fibroblasts exposed to bioactivehydrogels has been observed, while aggregation is not observed whenfibroblasts are exposed to the individual components of the bioactivehydrogel. Results from numerous (over fifty) controlled experiments haveshown that normal neonatal human skin fibroblasts form multi-cellaggregates when exposed to the complete thermoreversible hydrogelformulation at 37° C., while no such cell aggregating activity isdemonstrated using omission formulations in which the bioactivecopolymer is not formed. The aggregated cells form tightly apposed cellclusters with interdigitating cytoplasmic processes, while cells treatedwith formulations lacking the copolymer remain round and without surfaceprojections. As shown in FIG. 5, in a sample of human fibroblastsexposed to a bioactive hydrogel comprising dextran and gelatin, at least80% of the cells present were in an aggregated state while less than 20%of the cells present remained as single cells. The opposite effect wasobserved in samples where the human fibroblasts were exposed to collagenmonomer alone, carbohydrate alone, or were left untreated. In samplesexposed to collagen monomer alone, approximately 75% of the cellsremained in a single cell configuration while only about 25% of thecells were in an aggregated state. Nearly the same effect was observedin samples exposed to carbohydrate alone. In samples that were leftuntreated, approximately 60% of the cells remained in a single cellstate while only about 40% of the cells were in an aggregated state.

In each of the therapeutic uses outlined below, a therapeuticallyeffective amount of the matrix of the invention is used. Thetherapeutically effective dosage amount of any specific hydrogel matrixwill vary somewhat from matrix to matrix, patient to patient, use touse, and will depend upon factors such as the condition of the patient,the nature of the condition being treated, and the route of delivery.For example, a small dermal defect 1 cm in diameter and 0.5 cm deepwould require approximately 0.4 cm³ of stabilized cross-linked bioactivehydrogel to fill the void, stimulate vasculogenesis and tissueregeneration and have therapeutic efficacy. In contrast, a decubitusulcer 20 cm in diameter and 5 cm deep would require approximately 1600cm³ of stabilized cross-linked bioactive hydrogel to have similarefficacy. As a general proposition the amount of cross-linked bioactivematrix required for therapeutic efficacy will be from 0.1 to 2000 cm³,preferably from about 0.5 to 100 cm³.

In one aspect of the invention, the stabilized cross-linked bioactivehydrogel is used for site-specific tissue regeneration, includingvasculogenesis. It is known in the art to use intact collagen, gelatin,or dextran as a carrier to hold and deliver growth factors and the likein methods designed to promote tissue growth. (See, for example, Kawai,K. et al., “Accelerated tissue Regeneration Through Incorporation ofBasic Fibroblast Growth Factor-Impregnated Gelatin Microspheres intoArtificial Dermis” Biomaterials 21:489-499 (2000); and Wissink, M. J. B.et al., “Binding and Release of Basic Fibroblast Growth Factor fromHeparinized Collagen Matrices” Biomaterials 22:2291-2299 (2001)). Bycontrast, the intrinsic activity of the stabilized cross-linked hydrogelof the present invention is sufficient to elicit a specific sequence ofbiological responses, such as promoting tissue regeneration andvasculogenesis, without the addition of exogenous drugs or growthfactors. In fact, the cross-linked matrix of the invention can besubstantially free, even completely free, of exogenous drugs or growthfactors when used for vascularization or tissue regeneration. Thisintrinsically bioactive hydrogel, as a result of its unique structure,provides a cell attachment scaffold that modulates subsequent cellularactivity, such as tissue regeneration and vasculogenesis.

The intrinsic bioactivity of the stabilized cross-linked hydrogel isevident in its ability to promote vasculogenesis without the use ofadditional growth factors, such as basic fibroblast growth factor(bFGF). The cross-linked hydrogel may be used in vivo to facilitatevascularization in damaged tissue when placed at a vascular terminus andallowed to act as a vascular scaffold upon which new vascular tissue maygrow outward from the terminus. The vascular scaffold not only providesa support medium for the new vascular tissue, but it also performs thefunction of encouraging pro-lateral vessel growth while also providing asource of stabilization for the damaged area.

The stabilized cross-linked hydrogel behaves similarly when used inother aspects of tissue regeneration. The hydrogel provides a stabilizedstructural lattice that facilitates cell retention and multiplication inareas with tissue damage. This is due in part to the intrinsicbioactivity of the hydrogel, which furthers the regenerative process.

In another aspect of the invention, the stabilized cross-linked hydrogelis used as a bulking agent to provide increased dimensions to specifictissues requiring additional bulk, whether for aesthetic or functionalpurposes. Examples of such application include treatment of individualswith urinary incontinence and gastroesophageal reflux disease (GERD),problems commonly related to reduced sphincter tone. A sphincter is aringlike band of muscle fibers that acts to constrict a passage or closea natural orifice. Individuals with GERD generally exhibit multiplesymptoms stemming from gastric fluids that are allowed to pass from thestomach up into the esophagus because the sphincter at the base of theesophagus, which normally opens to allow materials to pass from theesophagus into the stomach and then closes, has reduced tone and failsto close completely. Individuals with GERD may be treated withmedications to reduce production of gastric fluids or hasten themovement of food from the stomach into the intestines; however, severecases often require corrective surgery, such as Nissen Fundoplicationwhere the upper portion of the stomach is wrapped around the loweresophagus to artificially tighten the esophageal sphincter. Similarly,urinary incontinence is often a result of reduced tone in the sphincterat the base of the bladder leading into the urethra and may requiremultiple therapies, including surgery.

The hydrogel of the present invention may be used to treat theseproblems, and others related to reduced sphincter tone. The hydrogel maybe injected into the sphincter either as a space-filling material or asa cell carrier to repopulate a local tissue defect thereby adding bulk,and allowing the sphincter to function normally again by using theincreased bulk to make up for the reduced tone, thereby allowing thesphincter to close completely. Such treatment is made possible due tothe increased stability of the cross-linked bioactive hydrogel, whichmaintains its structure at body temperatures and provides a biologicallycompatible, long-term solution that is much less invasive thanalternative treatments.

In a further aspect of the present invention, the cross-linked hydrogelmatrix may be combined with viable tissue cells for certain therapeuticuses. It is preferable, but not required, that the tissue cellsoriginate from the same type of tissue for which the hydrogel matrixwill be used therapeutically. The viable tissue cells can be derivedfrom autologous tissue sources, allogenic tissue sources, or xenogenictissue sources. The term “autologous” is meant to refer to tissue thatoriginates from the same host. The term “allogenic” is meant to refer totissue that originates from a source that is of the same species (i.e.,human) but of non-identical genetic composition. The term “xenogenic” ismeant to refer to tissue that originates from a species different fromthe host. Non-limiting examples of types of cells that can be used incombination with the hydrogel matrix include stem cells, bone cells,tenocytes, adipocytes, cardiomyocytes, hepatocytes, smooth muscle cells,endothelial cells, and the like. The tissue cells can be added to thehydrogel matrix prior to, during, or after cross-linking occurs.

One specific application for the hydrogel matrix combined with viabletissue cells is use of cells in the bulking agent application describedabove. Tissue cells that originate from the same type of tissuerequiring a bulking agent can be added to the cross-linked hydrogelmatrix prior to administration of the matrix to the anatomical siteneeding the bulking agent.

In another example, hepatocytes suspended in the matrix of the inventionprior to cross-linking are injected into a patient, and cross-linked insitu. The matrix provides a) a scaffold for the immobilized cells and b)a bioactive hydrogel for rapid vascularization at the site of implant.

In yet another example, the hydrogel matrix of the invention could beused as an ex vivo culture scaffold for the development of smalldiameter vascular grafts, valves, or other complex tissue-engineeredconstructs prior to implantation in a patient. In this case, thecross-linked hydrogel serves as an organizing template directing cellgrowth in vitro, and can be used to develop complex organ or tissuestructures through a sequence of culture steps.

In yet another aspect of the invention, the cross-linked hydrogel ismixed with other materials to form castable structures. For example, thecross-linked hydrogels can be mixed with osteoconductive orosteoinductive materials, such as calcium aluminate, hydroxyapatite,alumina, zirconia, aluminum silicates, calcium phosphate, bioactiveglass, ceramics, collagen, autologous bone, allogenic bone, xenogenicbone, coralline, or derivates or combinations thereof, or otherbiologically produced composite materials containing calcium orhydroxyapatite structural elements. The term “osteoconductive” is meantto refer to materials that facilitate blood vessel incursion and newbone formation into a defined passive trellis structure. The term“osteoinductive” is meant to refer to materials that lead to amitogenesis of undifferentiated perivascular mesenchymal cells leadingto the formation of osteoprogenitor cells (cells with the capacity toform new bone). By “alumina” is meant the commonly held definition ofmaterials comprised of the natural or synthetic oxide of aluminum, whichmay be exemplified in various forms, such as corundum. Bioactive glassesgenerally contain silicon dioxide (SiO₂) as a network former and arecharacterized by their ability to firmly attach to living tissue.Examples of bioactive glasses available commercially and theirmanufacturers include Bioglass® (American Biomaterials Corp., USA, 45%silica, 24% calcium oxide (CaO), 24.5% disodium oxide (Na₂O), and 6%pyrophosphate (P₂O₅)), Consil® (Xeipon Ltd., UK), NovaBone® (AmericanBiomaterials Corp.), Biogran® (Orthovita, USA), PerioGlass® (Block DrugCo., USA), and Ceravital® (E.Pfeil & H. Bromer, Germany). Corglaes®(Giltech Ltd., Ayr, UK) represents another family of bioactive glassescontaining pyrophosphate rather than silicon dioxide as a networkformer. These glasses contain 42-49 mole % of P₂O₅, the remainder as10-40 mole % as CaO and Na₂O.

The use of such materials as described above mixed with the stabilizedcross-linked hydrogel matrix of the present invention would be expectedto form castable cross-linked structures appropriate for bone repair andreconstruction as illustrated schematically in FIG. 6. As shown, theingredients for the cross-linked bioactive hydrogel matrix of theinvention are mixed in a vessel and allowed to react (i.e., cross-link)in the presence of finely divided ceramic powders, or otherosteoinductive material, to form a pourable paste as shown in Step 1.The paste is cast into a shaped mold and allowed to react and harden(Step 2). The final product is removed from the mold, and in thisinstance, is used as a dowel for bone repair (Step 3). This device orimplant is expected to induce vasculogenesis and hence betterintegration of the osteoinductive implant. Presumably, improvingvascular supply in large bones (e.g. femur) may increase marrowproduction and have a therapeutic effect beyond the simple improvementin bone density and health. Solid or semi-sold gels of this type couldbe utilized for tissue wounds, including bone fragment wounds ornon-healing fractures.

In another aspect of the invention, the stabilized cross-linked hydrogelis used as a wound healing device to protect open wounds during healingand also to promote healing by administration of the cross-linkedhydrogel to the wound. The individual abilities of collagen and gelatinto play a useful role in the area of wound coverings and wound healingare well documented. Collagen is known to perform the followingfunctions in wound healing: stop bleeding; help in wound debridement byattracting monocytes; provide a matrix for tissue and vascular growth;attract fibroblasts and help in directed migration of cells; bind withfibronectin which promotes cell binding; support cell growth,differentiation, and migration; and help in deposition of oriented andorganized fibers, which increases the integrity of tissue. Similarly,gelatin also effectuates wound healing and is known to stimulateactivation of macrophages and produce a high hemostatic effect. (See,for example, Hovig T. et al., “Platelet Adherence to Fibrin andCollagen” Journal Lab and Clin. Med. 71(1):29-39 (1968); Postlewaithe,A. E. et al., “Chemotactic Attraction of Human Fibroblasts to Type I,II, and III Collagens and collagen Derived Peptides” Proc. Natl. Acad.Science 177:64-65 (1978); Kleinman, H. K. et al., Role of CollagenousMatrices in the Adhesion and Growth of Cells” The Journal of CellBiology 88:473-485 (1981); Dunn, G. A. and Ebendal, T., “ContactGuidance on Oriented Collagen Gels” Exp. Cell Res. 111:475-479 (1978);Kleinman, H. K. et al., Interactions of Fibronectin with CollagenFibrils” Biochemistry 20:2325-2330 (1981); Morykwas, M. J. et al., “InVitro and In Vivo Testing of a Collagen Sheet to Support KeratinocyteGrowth for Use as a Burn Wound Covering” The Journal of Trauma29(8):1163-1167 (1976); Emerman, J. T. and Pitelka, D. R., “Maintenanceand Induction of Morphological Differentiation in Dissociated MammaryEpithelium on Floating Collagen Membranes” In Vitro 13(5):316-337(1977); Doillon, C. J. et al., “Fibroblast-Collagen Sponge Interactionsand Spatial Disposition of Newly Synthesized Collagen Fibers in Vitroand in Vivo” Scanning Electron Microscopy 3:1313-1320 (1984); and Hong,S. R. et al., “Study on Gelatin-Containing Artificial Skin IV: AComparative Study on the Effect of Antibiotic and EGF on CellProliferation During Epidermal Healing” Biomaterials 22:2777-2783(2001)).

It is believed that the stabilized cross-linked hydrogel of the presentinvention is useful as a wound healing device due to the intrinsicbioactivity of the material and the unique stereochemistry of themacromolecules in the presence of enhancing and strengthening polaramino acids. Several studies indicate the wound healing properties ofcollagen are attributable to its unique structure (see, Brass, L. F. andBensusan, H., “The Role of Quaternary Structure in the Platelet-CollagenInteraction” The Journal of Clinical Investigation 54:1480-1487 (1974);Jaffe, R. and Dykin, D., “Evidence for a Structural Requirement for theAggregation of Platelets by Collagen” Journal of Clinical Investigation53:875-883 (1974); Postlewaithe, A. E. and Kang, A. H., “Collagen andCollagen Peptide Induced Chemotaxis of Human Blood Monocytes” TheJournal of Experimental Medicine 143:1299-1307 (1976); and Reddi, A. H.,“Collagen and Cell Differentiation” In: Biochemistry of Collagen, NewYork; Plenum Press, 449-477 (1976)). Similarly, the hydrogel of thepresent invention demonstrates a unique activity as a wound healingdevice because of the unique structure of the hydrogel matrix, whichprovides a scaffold for cells and attracts tissue building componentsand factors necessary to promote wound healing. The rapid mechanicalintegration of the crosslinked hydrogels with the wound bed, the similarmechanical properties of the material, and its ability to act as apreferred cell attachment scaffold material in the wound bed contributeto the usefulness of the matrix as a wound healing device.

In another aspect, the stabilized cross-linked hydrogel may be used asan adhesive (i.e., tissue sealant) in wound repair. The use of adhesivesin wound repair is known in the art, and although such use has onlyrecently gained FDA approval in the United States, wound repairadhesives have been used extensively in Canada and Europe for more than20 years. Wound adhesives provide a popular alternative for woundclosure over standard methods, such as sutures, staples, and adhesivestrips, because they offer ease of use, decreased pain, reducedapplication time, and no follow-up for removal. The historically firstwound adhesive made available, and the one still used most often today,is a type of cyanoacrylate, or common household superglue. Earlier woundadhesives were composed of N-butyl cyanoacrylate, but the preferred formtoday is 2-octyl cyanoacrylate. The use of cyanoacrylate woundadhesives, however, has several drawbacks that limit its use, such as,allergic reactions, presence of residual solvents, and migration ofchemicals to other parts of the body. Further, cyanoacrylate adhesivesshould not be used in pregnant women or patients with a history ofperipheral vascular disease, diabetes mellitus, or prolongedcorticosteroid use, or on patients who have puncture wounds or bite orscratch wounds (animal or human in origin). Cyanoacrylate woundadhesives may only be used on the surface of the skin and on regularlyshaped wounds with even surfaces that are easily pushed back together.This is necessary to insure none of the cyanoacrylate touches raw skinor enters the wound because it may cause severe irritation and canactually function to impair epithelialization within the wound.

There are newer alternatives to cyanoacrylate wound adhesives, but manyof the alternatives possess additional drawbacks that complicate theirwidespread use. For example, adhesives composed of gelatin, resorcinol,and formaldehyde have been shown effective, but the toxicity andcarcinogenic effects of formaldehyde limit their use. Research has beenperformed indicating secretions from marine organisms, such as thoseused by barnacles to attach themselves to the hulls of ships, could beuseful wound adhesives, but the detailed genetic engineering used tocommercially produce the material has so far been found costprohibitive. Biological glues, such as fibrin glue, or hemostatic agentsare frequently used in cardiac or vascular surgeries to control diffusebleeding. One example of such a hemostatic sealant, FloSeal® (FloSealMatrix Hemostatic Sealant; Fusion Medical Technologies, Fremont,Calif.), is a combination of a cross-linked gelatin matrix and thrombin,which converts fibrinogen into fibrin monomers that polymerize to form afibrin clot. None of these alternatives, however, offer a viablealternative to cyanoacrylates as an easily used wound adhesive that maybe used in common practice.

The stabilized cross-linked hydrogel matrix of the present inventionexhibits properties that make it useful as a wound adhesive whileavoiding many of the drawbacks and contraindications associated withcyanoacrylates. The ability of the hydrogel to polymerize in situ hasthe effect of increasing cell-to-cell adhesion while simultaneouslyaccelerating vascularization and promoting wound healing. Thebiocompatibility of the hydrogel allows for its use in a wide array ofwounds, including situations where cyanoacrylates cannot be used, suchas open wounds, wounds with jagged edges, and wounds around mucousmembranes. In fact, the hydrogel of the present invention is mosteffective when introduced into the wound site as opposed to purelytopical use. When the nascent hydrogel mixture is placed into the wound,the in situ polymerization of the hydrogel acts to begin a cascade ofbiological interactions that seal the wound and facilitate the healingprocess. The active binding sites on the gelatin and dextranmacromolecules, in the presence of the added stabilizing and enhancingamino acids, not only cross-link with one another, but also form bondsto the native cells within the wound thereby forming a cross-linkedhydrogel matrix that acts to pull the wound surfaces toward the centralaxis of the wound and hold the wound edges together. In addition tofunctioning to hold the wound edges together, the hydrogel further actsto form a water-insoluble barrier between the wound site and theexterior elements and acts as a cellular scaffold to encourage tissueregeneration at the site.

There are many embodiments in which the hydrogel could be packaged anddelivered for use as a wound adhesive. For example, the reactive highmolecular weight components could be packaged in a dual chamberapparatus that keeps the components separated during storage and enablesthe components to be simultaneously expelled into the wound wherecross-linking could occur. Another contemplated embodiment involvespackaging the components in an apparatus with degradable membranesseparating the components Immediately prior to use, squeezing theapparatus would destroy the membranes allowing the components to mixproviding a limited window of time for application to the wound socross-linking could occur in situ. Various additional embodiments forpackaging and delivery of the hydrogel for use as a wound adhesive wouldbe readily apparent to one skilled in the art.

The bioactive cross-linked hydrogel matrix utilized in each of theembodiments described herein may be comprised solely of the two highmolecular weight components cross-linked to one another. Preferably,each of the embodiments described herein may incorporate additionalcomponents such as the enhancing agents utilized in the preferredembodiments described above. Table 1 below lists preferred componentspresent within the stabilized cross-linked hydrogel matrix of thepresent invention along with suitable concentrations as well aspreferred concentrations for each component. Note that theconcentrations listed in Table 1 for gelatin and dextran would also besuitable for alternative polyglycan and polypeptide components.

TABLE 1 Component Concentration Range Preferred Concentration L-glutamicacid 2 to 60 mM 15 mM L-lysine 0.5 to 30 mM 5.0 mM Arginine 1 to 40 mM10 mM Gelatin 0.01 to 40 mM 2 mM L-cysteine 5 to 500 μM 20 μM EDTA 0.01to 10 mM 4 mM Dextran 0.01 to 10 mM 0.1 mM (oxidized & native forms)

As noted above, the present invention provides numerous benefitsincluding eliciting vascularization at a localized site, modulatinglocalized wound healing response, and providing suitable means ofdeveloping a retrievable cell implantation device for cell-basedtherapeutics. Additional benefits may include the following: reducedscarring associated with degradation of bioerodible suture materials;improvement in the performance and long-term function of extravascularsensors such as glucose sensors routinely used for insulin deliverysystems; improvement in the rate of healing, durability, and mechanicalproperties around structural implants such as artificial joints andtendons; reduced pain and associated complications arising from postsurgical adhesions especially during abdominal or spinal injury; andimproved integration between natural tissues and implanted structures(i.e. teeth, porous hydroxyapatite or ceramic materials for bonerepair).

The cross-linked hydrogel matrix of the invention can be cross-linkedoutside the body and then implanted into a patient, or the hydrogelmatrix can be allowed to cross-link in situ. While the hydrogel isstable at body temperatures, the actual cross-linking of the gelatin anddextran may also take place at body temperatures. This characteristic isparticularly useful in view of the previously noted abilities of thecross-linked hydrogel to be used for tissue regeneration,vasculogenesis, as a bulking agent, and in other applications that wouldbe readily apparent to one skilled in the art. Irregular tissue defects,such as those common in chemical, thermal, or trauma wounds, whichrequire rapid healing, would also benefit from the ability to form insitu a bioactive hydrogel providing a cell attachment scaffold fortissue regeneration. An exemplary method for delivering the liquidcomponents of the hydrogel to the desired site for in situ formationinvolves using a multi-chamber syringe. The multi-chamber syringe may beattached to a multi-lumen catheter or needle such that the highmolecular weight components that form the cross-linked hydrogel do notinteract until injected into the site inside the body where the matrixis needed. Another contemplated method involves the use of themulti-chamber syringe with a single lumen catheter or needle containinga static mixing element where the components remain separated untilinjection into the site, but the high molecular weight componentsactually contact one another within the lumen of the catheter or needleduring injection into the specified site. Additional methods of deliveryof the hydrogel components for in situ formation would be readilyapparent to one skilled in the art. Typically, in the embodimentsdescribed above, one high molecular weight component, such as oxidizeddextran, would be placed in one chamber of the syringe and the otherhigh molecular weight component and additional enhancing agents would beplaced in a separate chamber.

EXPERIMENTAL

The present invention is more fully illustrated by the followingexamples, which are set forth to illustrate the present invention andare not to be construed as limiting thereof Unless otherwise indicated,all percentages refer to percentages by weight based on the total weightof the bioactive hydrogel matrix.

Example 1

20 g of dextran (MW 500,000 Da) was weighed into a tared beakercontaining 180 g phosphate-buffered saline. The dextran was dissolved,with constant stirring and 8 g sodium meta-periodate (available fromSigma, product number S1147) was added to the dissolved dextran. Thebeaker was wrapped in foil to prevent photo-catalyzed side-reactions,and placed in a refrigerator on a stirring plate for 12 hours at 5°C.±3° C. The beaker was removed, 50 mL ethylene glycol was added toconsume excess periodate, and the quenching reaction was allowed toproceed for 30 minutes at room temperature. The reaction mixture was pHadjusted to 7.5±0.5 with 0.1 N NaOH. The reaction products wereseparated using tangential flow filtration (Filtron Mini-Ultrasette PallFiltration Products, product number OS100C77). The solution mass wasreduced by half, and replaced with a 4-fold volume of phosphate bufferedsaline. The purified product was reduced to a final volume of 100 mL.The final product was filter sterilized as a 20% dextran solution, andstored frozen until use. Hydroxylamine titration showed that thisdextran was 20% oxidized.

A vial of a thermoreversible hydrogel matrix comprising gelatin anddextran and a vial of sterile filtered oxidized dextran were held at 39°C. for 30 minutes to melt the hydrogel and warm the oxidized dextran. Analiquot of 10 mL hydrogel was added to a 50 mL centrifuge tube, andrapidly mixed with 5 mL of oxidized dextran. The solution was cast into100 mm culture plate, and gently swirled to form a uniform film acrossthe bottom of the dish.

The reactive gel was allowed to cross-link at room temperature. The gelwas washed with Medium 199, at 37° C. by flooding the surface of the gelwith phenol red containing Medium 199. The Medium 199 overlay wasreplaced as required to maintain a neutral pH.

A tissue biopsy punch was used to produce 8 mm discs of cross-linkedgel. Individual discs were placed in a 15 mL centrifuge tube containing10 mL Medium 199 and were incubated at 37° C. for 2 weeks. Cross-linkedgels were insoluble at 37° C. and retained their initial shape.

Example 2

20 g of dextran (MW 500,000 Da) (available from Sigma, St. Louis, MO)was added to a tared beaker containing 200 mL of phosphate bufferedsaline (PBS) and stirred to form a uniform solution. A further 8 g ofsodium meta-periodate was added to the dextran solution, which waswrapped in foil, and allowed to stir overnight at 5° C.±3° C. Thereaction was quenched with 50 mL ethylene glycol, and the solution wasadjusted with 0.1 M NaOH to a pH of 7.5±0.5. The product was purifiedusing tangential filtration, and concentrated to a 20% dextran solution.Sterile filtered solutions were stored frozen until use. Hydroxylaminetitration showed that this dextran was 18% oxidized. Frozen samplesshowed no loss in oxidation levels after 8 months storage at 20° C.±5°C.

A series of thermoreversible hydrogel and oxidized dextran formulationswere prepared with fixed total gelatin concentration (12%) andincreasing concentrations of oxidized dextran. As illustrated in FIG. 7,the strength of the cast gels increased as the concentration of oxidizeddextran increased. Blends of fixed gelatin concentration and varyingoxidized dextran concentration were tested for resistance to compressionat two temperatures, 20° C. and 28° C. Gel strength increased withincreasing oxidized dextran content and decreasing temperature.

Example 3

20 g of dextran (MW 68,000 Da) (available from Sigma, St. Louis, Mo.)was added to a tared beaker containing 200 mL of phosphate bufferedsaline (PBS) and stirred to form a uniform solution. A further 8 g ofsodium meta-periodate was added to the dextran solution, which waswrapped in foil, and allowed to stir overnight at 5° C.±3° C. Thereaction was quenched with 50 mL ethylene glycol, and adjusted with 0.1M NaOH to a pH of 7.5±0.5. The product was purified using tangentialfiltration, and concentrated to a 20% dextran solution. Sterile filteredsolutions were stored frozen until use. Hydroxylamine titration showedthat this dextran was 14% oxidized.

A thermoreversible hydrogel comprising gelatin and dextran was meltedand added to several sets of mixtures of native and oxidized dextrans,mixed and cast into a T-25 culture flask. The concentration of oxidizeddextran in each sample ranged from about 3% to about 21%.

The cast gels were allowed to cure at 5° C.,±3° C., overnight, and werewashed extensively at 37° C. with phenol red containing Medium 199(available from Sigma Chemical Company, St. Louis, Mo.) until the nofurther change in pH was evident colorimetrically. The material wasrinsed extensively over four days with culture medium to neutralizeresidual acidic components.

Flasks containing 12% oxidized dextran were used for further cellculture studies. Normal neonatal human skin fibroblasts were provided in6 mL of serum-containing culture medium and allowed to interact with thematerial over an additional two weeks at 37° C. After 24 hours, cellsappeared to maintain normal health, showed attachment to the material byway of cytoplasmic processes, and also exhibited formation of multi-cellclusters. When observed 5 days later, one flask showed large cellaggregates that had formed in the culture and stellate cells in thelower layers of the culture where the large aggregates were attached tothe cross-linked hydrogel material. Over the subsequent week, theseaggregates continued to grow in size and appeared to contain healthycells. Cultures were imaged at this time, and the resulting figuresshowed the appearance of the cell aggregates rising above the materialsurface with elongated processes and individual cells connecting thestructure to the hydrogel substrate.

After approximately one month of exposure to the cross-linked material,cells were successfully dissociated from the hydrogel-containing flasksand replated onto standard tissue culture plastic surfaces, where theywere observed to grow readily and showed a morphology similar to that ofnormally cultured fibroblasts.

Example 4

A cross-linked hydrogel was prepared according to Example 1 above,wherein the hydrogel was comprised of 12% gelatin and 5% oxidizeddextran (MW 500,000 Da). After manufacture, 5 ml of the cross-linkedhydrogel was dispensed into a T-25 flask, to which was added 5 ml ofculture medium (IMDM containing 10% FBS). The cross-linked hydrogel wassolid at incubator temperature (37° C.). At 4 hours post-addition, theadded medium had undergone a color change from red to light yellow,indicating a change in solution pH toward acidic. The initial 5 ml ofculture medium was removed and a second 5 ml quantity was added to testthe buffering capacity of the medium. Three days later, the same colorchange was observed. The culture medium was again removed and replacedwith an additional 5 ml of culture medium. One day later, the there wasminimal color change indicating neutralization of acidic leachables fromthe material. On the same day, a population of human skin fibroblasts(product number CCD-1112Sk, American Type Culture Collection) wasdissociated and prepared for seeding into the flask. The cells wereseeded in fresh medium at a 1:6 dilution in 6 mL total volume, and theflasks were returned to the incubator. After one hour, the cells wereextending pseudopodia to connect with the cross-linked hydrogel, butwere not yet well attached. After approximately 4 additional hoursincubation, no additional attachment was observed. After one additionalday, approximately 20% of the cells appeared to be formingaggregate-like structures, with aggregates ranging in size from about 2to about 10 cells and attachment processes extending from the cells. Theexisting medium was poured off into a new T-25 flask and 3 mL of freshmedium was added to the culture. The following day, the original and thereplated cells were examined Both cell populations appeared rounded andunhealthy, and the transplanted cells had not attached to the new flasksurface.

Five days later (nine days from start of experiment), a third flask wasexamined, containing human skin fibroblasts, cross-linked hydrogel, andculture medium, which had been incubated undisturbed. This sampleexhibited large aggregates. When checked again the following day, theaggregated cell clusters had grown in size and resembled embryo-likestructures. Examination six days later revealed large multicellularstructures on top of the cross-linked hydrogel with some cellsapparently growing into the hydrogel at some sites.

Example 5

1.5 mL of a 0.5 mg/mL solution ofBis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), a crosslinkingagent, in dimethyl sulfoxide (DMSO), is added to a foil-wrapped vesselcontaining 15 mL of liquid thermoreversible hydrogel containing gelatinand dextran. Photoactivated non-specific cross-linking of thethermoreversible hydrogel occurs upon exposure of the reactive mixtureto long-wavelength light, such as that provided by continuous exposureto a 550-watt bulb (flood light used in photography). Longer exposuretimes demonstrated better cross-linking.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing descriptions andthe associated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosedherein and that modifications and other embodiments are intended to beincluded within the scope of the appended claims. Although specificterms are employed herein, they are used in a generic and descriptivesense only and not for purposes of limitation.

That which is claimed:
 1. A method for promoting tissue regeneration inmammals comprising the steps of: identifying a specific site in need oftissue regeneration; and administering a therapeutically effectiveamount of a cross-linked bioactive hydrogel matrix to the identifiedsite; wherein the bioactive hydrogel matrix comprises a polyglycancovalently cross-linked to a polypeptide and further comprising at leastone enhancing agent selected from the group consisting of polar aminoacids, divalent cation chelators, and combinations thereof; wherein thepolyglycan is a polysaccharide or a sulfated polysaccharide selectedfrom the group consisting of glycosaminoglycans, glucosaminoglycans,dextran, heparan, heparin, hyaluronic acid, alginate, agarose,carageenan, amylopectin, amylose, glycogen, starch, cellulose, chitin,heparan sulfate, chondroitin sulfate, dextran sulfate, dermatan sulfate,and keratan sulfate; wherein the polypeptide is a tissue-derivedpolypeptide selected from the group consisting of collagens, gelatins,keratin, decorin, aggrecan, glycoproteins, laminin, nidogen, fibulin,and fibrillin; and wherein the covalently cross-linked hydrogel matrixis a solid or semi-solid material at physiological temperature andphysiological pH.
 2. The method of claim 1, wherein said administeringstep comprises administering the hydrogel matrix at a vascular terminus,wherein the hydrogel matrix is positioned such that the matrix extendslaterally from the vascular terminus.
 3. The method of claim 1, whereinthe hydrogel matrix is cross-linked prior to administration.
 4. Themethod of claim 1, wherein the hydrogel matrix is cross-linked in situ.5. The method of claim 1, wherein the polyglycan has a molecular weightof about 2,000 to about 8,000,000 Da.
 6. The method of claim 1, whereinthe polypeptide is a tissue-derived polypeptide derived from extracts oftissue, wherein the tissue is selected from the group consisting ofsubmucosal tissues, arteries, vocal chords, pleura, trachea, bronchi,pulmonary alveolar septa, ligaments, auricular cartilage, abdominalfascia, liver, kidney, neurilemma, arachnoid, dura mater, and pia mater.7. The method of claim 1, wherein the polypeptide has a molecular weightof about 3,000 to about 3,000,000 Da.
 8. The method of claim 1, whereinthe polyglycan is dextran and the polypeptide is gelatin.
 9. The methodof claim 1, wherein the at least one enhancing agent comprises at leastone polar amino acid selected from the group consisting of tyrosine,cysteine, serine, threonine, asparagine, glutamine, aspartic acid,glutamic acid, arginine, lysine, histidine, and mixtures thereof. 10.The method of claim 1, wherein the at least one enhancing agentcomprises ethylenediaminetetraacetic acid or a salt thereof.
 11. Themethod of claim 1, wherein the tissue in need of regeneration isvascular tissue.
 12. The method of claim 1, wherein the tissue in needof regeneration is bone.
 13. The method of claim 12, wherein thebioactive hydrogel matrix further comprises an osteoconductive orosteoinductive material.
 14. The method of claim 13, wherein theosteoconductive or osteoinductive material is selected from the groupconsisting of calcium alum inate, hydroxyapatite, alumina, zirconia,aluminum silicates, calcium phosphate, bioactive glass, ceramics,collagen, autologous bone, allogenic bone, xenogenic bone, coralline,and derivates or combinations thereof.
 15. A method for promoting tissueregeneration in mammals comprising the steps of: identifying a specificsite in need of tissue regeneration; and administering a therapeuticallyeffective amount of a cross-linked bioactive hydrogel matrix to theidentified site; wherein the bioactive hydrogel matrix comprises apolyglycan covalently cross-linked to a polypeptide between functionalgroups on the polypeptide and functional groups on the polyglycan orwith a bifunctional cross-linker molecule, and wherein the bioactivehydrogel matrix further comprises at least one enhancing agent selectedfrom the group consisting of polar amino acids, divalent cationchelators, and combinations thereof.
 16. The method of claim 15, whereinthe tissue in need of regeneration is vascular tissue.
 17. The method ofclaim 15, wherein the tissue in need of regeneration is bone.
 18. Themethod of claim 17, wherein the bioactive hydrogel matrix furthercomprises an osteoconductive or osteoinductive material.
 19. The methodof claim 18, wherein the osteoconductive or osteoinductive material isselected from the group consisting of calcium aluminate, hydroxyapatite,alumina, zirconia, aluminum silicates, calcium phosphate, bioactiveglass, ceramics, collagen, autologous bone, allogenic bone, xenogenicbone, coralline, and combinations thereof.